Purification and properties of two enzymes catalyzing galactose transfer to GM2 ganglioside from rat liver Golgi

An enzyme activity which catalyzed the transfer of galactose from UDP-galactose to GM2 ganglioside was demonstrated in rat liver homogenate and enriched 38-fold in specific activity by preparation of Golgi membranes. This activity could be solubilized from Golgi membranes by sonication and extraction with 1% Triton X-100. The solubilized activity catalyzed the formation of GM1 ganglioside and was completely dependent upon the addition of acceptor. The rate of galactose incorporation was constant for up to 5 h at 30 degrees C. This enzyme activity was further purified by gel filtration on Sepharose CL-6B and ion exchange chromatography on DEAE-Sepharose. The elution position on gel filtration corresponded to a molecular weight for the enzyme of 38,000 which was in good agreement with that obtained by sedimentation velocity studies. Ion exchange chromatography resolved GM2 ganglioside galactosyltransferase into two species. The more basic enzyme (I) comprising 28% of the recovered activity was not retarded by the column, whereas enzyme II was eluted from the resin following the application of a salt gradient. Net purification was 120- to 140-fold for each enzyme with a total recovery of 42%. Unlike the activity in the Golgi extract, the purified enzymes I and II were labile to freezing and could be stored at -20 degrees C only in the presence of 50% glycerol. Both enzymes I and II had similar molecular weights and Michaelis constants and both had a strict requirement for Mn2+. Properties which distinguish the two enzymes included pH optima (enzyme I 7.0, enzyme II 6.0) and surfactant requirements. Neither enzyme was active following removal of Triton X-100 from the preparation. Among a series of glycolipids tested for ability to serve as substrates for galactose transfer only GM2 and asialo-GM2 ganglioside served as acceptors.     

One-step purification of chitinase from Serratia marcescens NK1, a soil isolate

AIMS: A simple single step technique of gel filtration was developed for the purification of chitinase from Serratia marcescens NK1. METHODS AND RESULTS: Chitinase from Ser. marcescens NK1 was purified to homogeneity by gel filtration chromatography with 9.2% recovery. The enzyme had a pH optimum of 6.2 and a temperature optimum of 47 degrees C. It was stable in a wide pH range of 3.0 to 10.0, retaining 60% activity at pH 3.0 and 65% activity at pH 10.5. It retained 70% activity at 28 degrees C after 72 h and nearly 50% activity at 50 degrees C up to 24 h. CONCLUSION: The chitinase from Ser. marcescens NK1 can be efficiently purified in a single step by gel filtration chromatography. The chitinase of Ser. marcescens NK1, a soil isolate, is highly stable and as active as that of other reported isolates of Ser. marcescens. SIGNIFICANCE AND IMPACT OF THE STUDY: This purification scheme is advantageous because of its simplicity and can therefore be applied for the purification of other enzymes. The yield is sufficient for initial characterization studies of the enzyme, and an improved resolution can be obtained if the chromatography is done under fast flow systems.     

Pine seed ribonucleases. I. Preparation and properties.

The enzyme preparation, about 100 fold purified, shows optimal activity at pH 4.8 and 5.9. This activity lessens rapidly at 40-60 degrees C, and retains 20% at 100 degrees C. These results of heat stability and optimal pH might suggest that the enzyme preparation contains two enzymes. The use of gel filtration clearly shows that pine RNAses are endonuclease.     

Membrane-bound 'synthetic lipase' specifically cultured under solid-state fermentation and submerged fermentation by Rhizopus chinensis: a comparative investigation

Rhizopus chinensis was able to produce synthetic lipases under both solid-state and submerged fermentations. These lipases were extracted from cell membrane using Triton X-100, and purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction chromatography and gel filtration chromatography. Judging from SDS-PAGE, the specific synthetic lipases associated with SSF (named as SSL) and SmF (named as SML) were different in the apparent molecular mass (62 and 40kDa). In term of hydrolytic activity, both enzymes exhibited maximum values at pH 8.0 and 40 degrees C; SSL appeared to be more pH tolerant and thermostable than SML. PMSF negligibly affected SSL but strongly reduced the activity of SML. Both enzymes showed clear preference for long-chained p-nitrophenyl esters, yielding maximum activity towards p-nitrophenyl palmitate (with SSL) and p-nitrophenyl laurate (with SML). In term of synthetic activity, lyophilized enzymes gave the highest values both at 30 degrees C, but at different pH memories (7.5 for SSL and 6.5 for SML). Most of ethyl esters synthesized by the two enzymes achieved good yields (>90%), and tetradecanoic acid and laurate acid separately served as the best acyl donors.     

Properties of soluble alpha-chymotrypsin in neat glycerol and water

UV scanning of alpha-chymotrypsin dissolved in neat glycerol and water showed no significant differences in its spectra at pH 7.8. Fluorescence scanning revealed a strong dependence on pH values (between 5.9 to 10.5) of the maximum wavelength emission in water and no pH-dependence in 99% glycerol supplemented with 1% of appropriate buffers. The profile of alpha-chymotrypsin activity dissolved in water-glycerol mixtures with phenyl acetate as substrate displayed two maximum: highest peak was found at 100% water, and the second one was observed in 99% glycerol concentration with about 40% of the relative activity. Optimum pH of the soluble alpha-chymotrypsin in glycerol showed a displacement of 1 pH/U towards the alkaline side compared to water at pH 8.0. Kinetic and thermodynamic analysis using kinetic measurements of the thermal stability of alpha-chymotrypsin showed a higher inactivation rate in neat glycerol as compared to water in 30 to 45 degrees C range, however, when temperature increases enzyme stability in glycerol is better than water. Thermostability of trypsin and alpha-chymotrypsin dissolved in glycerol at 100 degrees C showed a half reaction time of approximately 7 and 20 h, respectively, and less than 1 minute in aqueous buffer for both enzymes.     

Characterization of two acid proteinases found in rabbit skin homografts.

Two types of acid proteinase activity found in rabbit skin homografts were characterized by studying the effect of temperature, pH and polyacrylamide-gel electrophoresis. Their chromatographic behaviour was characterized on DEAE-cellulose, Sephadex G-75, G-100 and G-200, and their molecular weights were estimated by gel filtration. One of the acid proteinases in the homograft resembled cathepsin D (EC 3.4.23.5) of normal skin. The other acid proteinase differed from cathepsin D with respect to heat inactivation, pH optimum and molecular weight; it was not inactivated on heating at 60 degrees C for 60 min, its pH optimum was 2.5 and its molecular weight measured by Sephadex G-100 chromatography was 100 000. In all these respects, the heat-stable proteinase resembles cathepsin E (EC 3.4.23.5) of rabbit polymorphonuclear leucocytes.     

Studies on Lipase in Rat Brain

Lipase activity was measured in homogenates of rat cerebral hemispheres using radioactive glycerol trioleate emulsified with Triton X-100 as substrate. The labeled oleic acid was separated from the ester with a methanol-chloroform-heptane mixture. Under these assay conditions, the activity showed pH optima at about 5.5 and 7.5. The final products of these lipase activities were suggested to be free fatty acid and glycerol.     

Enzymes of carbohydrate metabolism of mycelial fungi from marine environments. beta-1,3-glucanase of the marine fungus Chaetomium indicum

Thirty samples of fungi belonging to 17 species living in marine environments were studied for their ability to produce extracellular enzymes. In the culture fluids, a variety of glycosidases (beta-glucosidases, N-acetyl-beta-glucosaminidase, beta-galactosidases, and alpha-mannosidases) and glucanases (amylases and beta-1,3-glucanases) were found. Several cultures were found that could be used as efficient producers of either individual enzymes or a whole complement of enzymes degrading carbohydrate-containing compounds. Optimal growth conditions for the fungus Chaetomium indicum and beta-1,3-glucanase biosynthesis were developed. beta-1,3-Glucanase was isolated by a combination of ion-exchange chromatography, ultrafiltration, and gel chromatography. The molecular mass of the enzyme determined by gel-filtration was 54 kD. The enzyme was stable at temperatures below 50 degrees C, had a temperature optimum for activity at 60 degrees C, and retained activity between pH 4.5 and 7.5. The pH dependence of the beta-1, 3-glucanase activity showed two maxima, at pH 4.4 and 5.6; this suggested the existence of two forms of the enzyme. Analysis of the products of enzymatic hydrolysis of laminaran, transglycosylating ability, and the effect of a specific natural inhibitor indicates that both forms are exo-beta-1,3-glucanases.     

Purification and properties of bacteriolytic enzymes from Bacillus licheniformis YS-1005 against Streptococcus mutans

To find a novel lytic enzyme against cariogenic Streptococci, strains showing strong lytic activity have been screened from soil using Streptococcus mutans. A strain identified as Bacillus licheniformis secreted two kinds of lytic enzymes, which were purified by methanol precipitation, CM-cellulose chromatography, gel filtration, and hydroxyapatite chromatography. The molecular weights of these two enzymes, L27 and L45, were 27,000 and 45,000, respectively. Optimum pH and temperature of both enzymes for lytic activity were pH 8 and 37 degrees C. L27 and L45 digest the peptide linkage between L-Ala and D-Glu in peptidoglycan of Streptococcus mutans. The lytic activity was highly specific for Streptococcus mutans, suggesting their potential use as a dental care product.     

Purification and Characterization of Two Highly Thermophilic Alkaline Lipases from Thermosyntropha lipolytica

Two thermostable lipases were isolated and characterized from Thermosyntropha lipolytica DSM 11003, an anaerobic, thermophilic, alkali-tolerant bacterium which grows syntrophically with methanogens on lipids such as olive oil, utilizing only the liberated fatty acid moieties but not the glycerol. Lipases LipA and LipB were purified from culture supernatants to gel electrophoretic homogeneity by ammonium sulfate precipitation and hydrophobic interaction column chromatography. The apparent molecular masses of LipA and LipB determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 50 and 57 kDa, respectively. The temperature for maximal activity of LipA and LipB was around 96°C, which is, so far as is known, the highest temperature for maximal activity among lipases, and the pH optima for growth determined at 25°C (pH^25°C optima) were 9.4 and 9.6, respectively. LipA and LipB at 100°C and pH^25°C 8.0 retained 50% activity after 6 and 2 h of incubation, respectively. Both enzymes exhibited high activity with long-chain fatty acid glycerides, yielding maximum activity with trioleate (C_18:1) and, among the p-nitrophenyl esters, with p-nitrophenyl laurate. Hydrolysis of glycerol ester bonds occurred at positions 1 and 3. The activities of both lipases were totally inhibited by 10 mM phenylmethylsulfonyl fluoride and 10 mM EDTA. Metal analysis indicated that both LipA and LipB contain 1 Ca²⁺ and one Mn²⁺ ion per monomeric enzyme unit. The addition of 1 mM MnCl₂ to dialyzed enzyme preparations enhanced the activities at 96°C of both LipA and LipB by threefold and increased the durations of their thermal stability at 60°C and 75°C, respectively, by 4 h.     

Characterization of turkey pancreatic lipase

Turkey pancreatic lipase (TPL) was purified from delipidated pancreases. Pure TPL (glycerol ester hydrolase, EC 3.1.1.3) was obtained after ammonium sulfate fractionation, Sephacryl S-200 gel filtration, anion exchange chromatography (DEAE-Sepharose) and size exclusion column using high performance liquid chromatography system (HPLC). The pure lipase, which is not a glycoprotein, was presented as a monomer having a molecular mass of about 45 kDa. The lipase activity was maximal at pH 8.5 and 37 degrees C. TPL hydrolyses the long chains triacylglycerols more efficiently than the short ones. A specific activity of 4300 U/mg was measured on triolein as substrate at 37 degrees C and at pH 8.5 in the presence of colipase and 4 mM NaTDC. This enzyme presents the interfacial activation when using tripropionin as substrate. TPL was inactivated when the enzyme was incubated at 65 degrees C or at pH less than 5. Natural detergent (NaTDC), synthetic detergent (Tween-20) or amphipatic protein (beta-lactoglobulin A) act as potent inhibitors of TPL activity. To restore the lipase activity inhibited by NaTDC, colipase should be added to the hydrolysis system. When lipase is inhibited by synthetic detergent or protein, simultaneous addition of colipase and NaTDC was required to restore the TPL activity. The first 22 N-terminal amino acid residues were sequenced. This sequence was similar to those of mammal's pancreatic lipases. The biochemical properties of pancreatic lipase isolated from bird are similar to those of mammals.     

Purification and characterization of a new xylanase (APX-II) from the fungus Aureobasidium pullulans Y-2311-1.

Aureobasidium pullulans Y-2311-1 produced four major xylanases (EC 3.2.1.8) with pI values of 4.0, 7.3, 7.9, and 9.4 as revealed by isoelectric focusing and zymogram analysis when grown for 4 days on 1.0% oat spelt xylan. The enzyme with a pI of 9.4 was purified by ammonium sulfate precipitation, chromatography on a DEAE-Sephadex A-50 column, and gel filtration with a Sephadex G-75 column. The enzyme had a mass of about 25 kDa as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. The purified enzyme had a Km of 7.6 mg . ml(-1) and Vmax of 2,650 micromol . min(-1) . mg(-1) for birchwood xylan at 28 degrees C and pH 4.5. It lacked activity towards carboxymethylcellulose, cellobiose, starch, mannan, p-nitrophenyl (pNP)-beta-D-xylopyranoside, pNP-beta-D-glucopyranoside, pNP-alpha-D-glucopyranoside, pNP-beta-D-cellobioside, pNP-beta-D-fucopyranoside, or pNP-alpha-D-galactopyranoside. The predominant end products of birchwood xylan or xylohexaose hydrolysis were xylobiose and xylose. The enzyme had the highest activity of pH 4.8 and 54 degrees C. Sixty percent of the activity remained after the enzyme had been incubated at 55 degrees C and pH 4.5 for 30 min. The sequence of the first 68 amino acid residues at the amino terminus showed homology to those of several other xylonases. Immunoblot analysis with antiserum raised against the purified xylanase revealed that two immunologically related polypeptides of 25 and 22 kDa were produced in A. pullulans cultures containing oat spelt xylan or xylose as carbon sources but not in cultures containing glycerol or glucose.     

Various properties of cathepsin G and elastase from swine peripheral blood neutrophils

Using gel filtration through Sephadex G-100 and bioaffinity chromatography on contrical-Sepharose, cathepsin G and elastase were isolated from pig peripheral blood neutrophil granules and purified to homogeneity. Both enzymes hydrolyzed the total histone from calf thymus as well as synthetic substrates--tert-butoxy-L-alanine p-nitrophenyl ester (elastase) and benzoyltyrosine ethyl ester (cathepsin G). The use of natural and synthetic protease inhibitors showed that both enzymes were related to the group of serine proteases. The molecular mass of the cathepsin G subunit as determined by SDS polyacrylamide gel electrophoresis is 28-29 kD, that of elastase--30-31 kD. The pH optima for the hydrolysis of proteinaceous and synthetic substrates for cathepsin G and elastase are 8.0-8.5 and 7.0-7.5, respectively. The isoelectric points for elastase and cathepsin G are 9.7-10.0 and greater than 10, respectively; the temperature optima--30-40 degrees C and 50-60 degrees C, respectively. The amino acid composition of the two enzymes from pig granulocytes revealed a high content of arginine and was similar to that of human granulocytes.     

Lipase activity in the human aorta

The hydrolysis of triglycerides by grossly normal male human aortas has been studied in vitro. The tissue contains an acid lipase (pH optimum, 5.4) and an alkaline lipase (pH optimum, 8.8). Both lipases catalyze the hydrolysis of saturated triglycerides; the rate decreases with increasing fatty acyl chain from C(10) to C(18). Glycerol trioleate, trilinoleate, and trilinolenate are hydrolyzed at similar rates. Alkaline lipase is inhibited about 50% at 7.2 mm glycerol trioleate, while acid lipase is unaffected at this concentration. Both lipases are activated by Ca(++) ions. The acid lipase is easily inactivated by deionized water used either as a homogenizing or dialyzing medium. Acid lipase is strongly inhibited by BSA, sodium deoxycholate, and sodium taurocholate; alkaline lipase is unaffected by BSA and is activated about twofold by bile salts. The products of hydrolysis of glycerol trioleate by aortic lipases are predominantly oleic acid and glycerol 1,2-dioleate with a small accumulation of glycerol monooleate. The aortic preparations appear to contain inhibitors for both the acid and alkaline lipase. The substance which inhibits alkaline lipase also inhibits pancreatic lipase; it is heat-stable and dialyzable. The inhibitor of the acid lipase is also heat-stable but is nondialyzable.     

A bifunctional beta-xylosidase-xylose isomerase from Streptomyces sp. EC 10

beta-Xylosidase (1,4-beta-D-xylan xylohydrolase EC 3.2.1.37) and xylose isomerase (D-xylose ketol-isomerase EC 5.3.1.5) produced by Streptomyces sp. strain EC 10, were cell-bound enzymes induced by xylan, straw, and xylose. Enzyme production was subjected to a form of carbon catabolite repression by glycerol. beta-Xylosidase and xylose isomerase copurified strictly, and the preparation was found homogeneous by gel electrophoresis after successive chromatography on DEAE-Sephacel and gel filtration on Biogel A. Streptomyces sp. produced apparently a bifunctional beta-xylosidase-xylose isomerase enzyme. The molecular weight of the enzyme was measured to be 163,000 by gel filtration and 42,000 by SDS-PAGE, indicating that the enzyme behaved as a tetramer of identical subunits. The Streptomyces sp. beta-xylosidase was a typical glycosidase acting as an exoenzyme on xylooligosaccharides, and working optimally at pH 7.5 and 45 degrees C. The xylose isomerase optimal temperature was 70 degrees C and maximal activity was observed in a broad range pH (5-8). Enhanced saccharification of arabinoxylan caused by the addition of the enzyme to endoxylanase suggested a cooperative enzyme action. The first 35 amino acids of the N-terminal sequence of the enzyme showed strong analogies with N-terminal sequences of xylose isomerase produced by other microorganisms but not with other published N-terminal sequences of beta-xylosidases.     

Purification and Characterization of Extracellular Amylolytic Enzymes from the Yeast Filobasidium capsuligenum

The extracellular amylolytic system of Filobasidium capsuligenum consisted of an alpha-amylase (1,4-alpha-d-glucan glucanhydrolase, EC 3.2.1.1) and two forms of glucoamylase (1,4-alpha-d-glucan glucohydrolase, EC 3.2.1.3). The enzymes were purified by ammonium sulfate fractionation, repeated ion-exchange chromatography (DEAE-Sephadex A-50), and gel filtration (Sephadex G-25, Sephadex G-100 sf). alpha-Amylase had an optimum pH of 5.6 and an optimum temperature of 50 degrees C but was rapidly inactivated at higher temperature. The molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 64,000. An acarbose concentration of 20 mug/ml was required for 50% inhibition of the alpha-amylase. Both glucoamylases are glycoproteins of identical molecular weight (60,000) and produce only glucose by exohydrolysis. The debranching activity of the glucoamylases was evidenced with substrates containing alpha-1,6 linkages. The pH optima were 5.0 to 5.6 for glucoamylase I and 4.8 to 5.3 for glucoamylase II. Glucoamylase I had a higher optimum temperature (55 degrees C) than glucoamylase II (50 degrees C) and was also more resistant to thermal inactivation. Only low acarbose concentrations (<0.1 mug/ml) were required to reduce the activity of the glucoamylases by 50%.     

Some properties of proteolytic enzymes from Tribolium confusum

The digestive tract of Tribolium confusum Duv. larvae was studied for proteolytic enzymes properties. The pH optima are determined for the enzymes effect on various substrates. Proteases were partially purified by gel chromatography on Sephadex G = 100 and investigated for thyol compound influence on their activity. The activity of the enzymes is shown to increase considerably with addition of cystein, glutathione, 2-mercaptoethanol. dithiotreitol and EDTA. Dithiotreitol produces the strongest restoring effect and in concentration of 10(-6) M it activates the enzyme almost twice. Storage for 48 h at 4 degrees C induced a 2.5-fold decrease in the proteolytic enzyme activity; SH-groups in the catalytic action of enzymic solutions is shown. The maximum proteolytic activity is found in extracts from 14-day insects.     

Purification and investigation of some kinetic properties of glucose-6-phosphate dehydrogenase from parsley (Petroselinum hortense) leaves

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.     

Purification and Properties of Pyranose Oxidase from Coriolus versicolor

Coriolus versicolor KY2912 grown on a medium containing glucose, sucrose or glycerol produced pyranose oxidase. Pyranose oxidase (glucose-2-oxidase) was purified by HPA-75 chromatography, Sepharose 4B and Sephadex G-100 gel filtration, and hydroxyapatite chromatography. The purified enzyme preparation showed a single protein band on acrylamide gel electrophoresis. The highest activity was obtained when D-glucose was employed as substrate and molecular oxygen as electron acceptor. The enzyme was most active at pH 6.2 and 50°C, stable in the pH region between 5.0 and 7.4, and the activity was completely lost above 70°C. The activity was inhibited by Ag⁺ , Cu²⁺ and PCMB. The enzyme contained FAD covalently bound to the polypeptide chain. The enzyme consisted of identical subunits with a molecular weight of 68,000, and showed a total molecular weight of 220,000.     

Purification and some properties of a thermostable DNA polymerase from a Thermotoga species

A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5-8. When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.